Pharmaceutical/ cosmetic composition comprising hyaluronic acid and treatment of dermatological conditions therewith

ABSTRACT

Pharmaceutical/cosmetic compositions containing a dermatologically effective amount of hyaluronic acid, at least one retinoid and/or salt and/or derivative thereof, at least one oligosaccharide and at least one inhibitor of hyaluronic acid degradation, formulated into a physiologically acceptable medium therefor, are useful for the treatment of wrinkles, fine lines, fibroblast depletions and scars.

CROSS-REFERENCE TO EARLIER APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 12/213,690, filed Jun. 23, 2008, which is a continuation of PCT/FR 2006/051391, filed Dec. 19, 2006 and designating the United States (published in the French language on Jul. 5, 2007 as WO 2007/074288 A1; the title and abstract were also published in English), which claims priority under 35 U.S.C. §119 of FR 0513055, filed Dec. 21, 2005, each hereby expressly incorporated by reference in its entirety and each assigned to the assignee hereof.

BACKGROUND OF THE INVENTION

1. Technical Field of the Invention

The present invention relates to preparations for topical and/or parenteral administration, comprising hyaluronic acid formulated into a physiologically acceptable medium, to processes for the production of such preparations, and to uses thereof as a medicament, such preparations being especially useful for the treatment of dermatological conditions and afflictions, in particular for the treatment of wrinkles, fine lines, fibroblast depletions and any scars.

2. Description of Background and/or Related and/or Prior Art

Skin aging is one of the most visible modifications of the process of senescence. In addition, the skin is exposed to many factors that accelerate this physiological process. A distinction can be made from two different types of skin aging. Firstly, intrinsic aging, that it is easier to evaluate on areas which are not normally exposed to the sun and, secondly, extrinsic aging, brought about by the interaction of environmental factors, in particular UV rays. These environmental factors have a much more marked effect on the parts of the body exposed to the sun, especially in individuals of light phototype. This is then also referred to as actinic aging. Other factors, such as dietary habits, smoking, excessive alcohol consumption, chronic diseases and endocrine gland dysfunctions, also contribute to this aging.

During intrinsic skin aging, the horny layer is relatively unmodified. The epidermis is atrophic and the dermal-epidermal junction is flattened, such that the adhesion to the dermis is weaker, facilitating the formation of bubbles. The thickness of the dermis is clearly reduced; there are fewer blood vessels. Fewer fibroblasts are also observed and their biosynthetic and proliferative capacities are reduced. The elastic fibers first undergo modifications, and subsequently disappear.

As regards extrinsic aging, an irregular, sometimes atrophic, sometimes hyperplasic, epidermis is observed, with signs of disorganization and of dysplasia. There are more melanocytes in certain areas, and fewer in others. The distribution of melanin in the epidermis is also irregular, subsequent to melanosome transfer problems. The number of Langerhans cells decreases. The small blood vessels are first dilated, and then become thinner and atrophy.

Wrinkles are the most visible signs of aging. A distinction can be made from several types, in particular superficial and deep wrinkles. Deep wrinkles are thought to be due to dermo-hypodermal modifications, whereas superficial wrinkles could be explained by dermal and possibly epidermal modifications. Wrinkles are especially due to the loss of elasticity of the skin. The effect on the subepidermal elastic network gives rise to superficial laxity of the aged skin and folding of its surface. The destruction of the elastic fibers in the reticular dermis is responsible for the loss of elasticity and of the skin's ability to return to its shape after stretching. A suitable treatment will be possible according to the type, the intensity and the topography.

The treatment of unattractive skin modifications related to aging has made enormous progress over the past few years.

A relatively large number of natural or synthetic substances have already been described as dermal implants, i.e., as substances injected directly into the skin, in order to remedy skin alterations resulting from aging, traumas or diseases.

Other therapeutic alternatives for these applications are in particular the local injection of botulinum toxin (Botox®) or the use of laser techniques. These various types of treatment are not exclusive and a combination thereof has even been recommended. Among the natural substances of human origin, collagen and hyaluronic acid are those which form the basis of the majority of products available on the market.

Hyaluronic acid is a ubiquitous natural polysaccharide which exists in the same form from the simplest bacterium to humans. It is a polymer of disaccharides which are themselves composed of D-glucuronic acid and N-acetylglucosamine, linked to one another by alternating beta-1,4 and beta-1,3 glycosidic linkages. The polymers of this recurring unit may be from 102 and 104 kDa in size, in vivo. Hyaluronic acid represents in particular a natural constituent of the dermis, where it plays an important role in the hydration and elasticity of the skin. However, it decreases in amount and in quality with age, leading to drying out of the skin, which becomes wrinkled. It is highly water-soluble and forms high-viscosity solutions in water. Because of these specific properties, hyaluronic acid is among the pharmaceutical products most commonly used.

However, in humans, hyaluronic acid is very rapidly eliminated from the plasma by degradation. Its plasma half-life after intravenous injection is very short, from 2.5 to.5 minutes, whereas in the skin, its half-life is from 0.5 to 2 days depending on its concentration. Its excretion in the urine is low, less than 1% of total clearance. In rabbits, the rate of elimination, in the skin, has been measured (Reed R K, Laurent U B, Fraser J R, Laurent T C. Removal rate of [3H]hyaluronan injected subcutaneously in rabbits, Am. J. Physiol., 1990 August; 259 (2 Pt 2): H532-5). It is non-exponential with a half-life of 0.5 to 1 day when its concentration is 5 mg/ml.

The tolerance of hyaluronic acid is very good and no immunogenicity has been associated with this substance. A very low incidence of side effects is thus observed.

The use of hyaluronic acid, alone or in combination, has thus been described for several medical applications, such as, for example, the treatment of osteoarthritis and also rheumatoid arthritis. Injectable compositions such as, for example, hyaluronic acid alone, collagen alone or the combination of “hyaluronic acid and collagen” have also already been employed in repair surgery, in the context of the treatment by filling of wrinkles, fine lines, fibroblast depletions and any scars.

Currently, many dermal implants are used but none has yet been considered to be ideal in the context of a safe and healthy tissue augmentation (Naoum C, Dasiou-Plakida D. Dermal filler materials and botulin toxin, Int. J. Dermatol., 2001 October; 40(10): 609-21).

However, because the bioavailability of hyaluronic acid is too low after injection and its injection frequency is too high, it cannot be used as such.

Of course, there has been an effort to develop compositions based on hyaluronic acid having a very good bioavailability and capable of more successfully withstanding the action of degradation enzymes. This makes it possible, in particular, to space out the procedures and to reduce the number thereof.

These compositions employed as a dermal implant are all composed of stabilized hyaluronic acid and a large number of these comprise hyaluronic acid that has been chemically modified for this purpose. In addition, the hyaluronic acid included in these products is predominantly of nonhuman origin, for instance of avian or bacterial origin.

Numerous chemically modified hyaluronic acid derivatives in the form, in particular, of esters, amides and also derivatives having “intra- and/or interchain bridges” (crosslinked), are thus found in these compositions.

However, these modifications affect the physicochemical characteristics and the biological properties of hyaluronic acid, and also its outcome after administration. These structural modifications of hyaluronic acid can lead to inflammatory reactions, as reported by Sopaar C N S, Patrinely J R Ophthalmic plastic and reconstructive surgery 2005 March; 21(2): 151-53.

Furthermore, the bioavailability of these hyaluronic acid derivatives, although better than that of natural hyaluronic acid, still remains too short.

SUMMARY OF THE INVENTION

Novel preparations comprising hyaluronic acid have now been developed having a better bioavailability while at the same time conserving the physicochemical characteristics and biological properties thereof, as well as a process for the formulation of such preparations.

Thus, the present invention features pharmaceutical or cosmetic compositions or preparations, in particular for topical and/or parenteral administration, comprising, formulated into a physiologically acceptable medium, hyaluronic acid, and also:

at least one retinoid and/or salts thereof and/or derivatives thereof,

at least one oligosaccharide, and

at least one inhibitor of hyaluronic acid degradation.

The present invention also features a process for the production of a pharmaceutical or cosmetic composition or preparation for topical and/or parenteral application, comprising, in a physiologically acceptable medium, hyaluronic acid, and at least one retinoid and/or salts thereof and/or derivatives thereof, at least one oligosaccharide and at least one inhibitor of hyaluronic acid degradation, which comprises the step of mixing an effective amount of hyaluronic acid with at least one retinoid and/or salts thereof and/or derivatives thereof, at least one oligosaccharide and at least one inhibitor of hyaluronic acid degradation. Preferably, the process according to the invention also comprises a step of preparing a physiologically acceptable medium, in which the active agents are mixed.

Finally, this invention features administration of the subject preparations as medicaments for the treatment and/or prevention of dermatological conditions/afflictions, whether comprising a regime or regimen.

When a pharmaceutical or cosmetic composition or preparation for topical and/or parenteral application comprises, formulated into a physiologically acceptable medium, hyaluronic acid, and at least one retinoid and/or salts thereof and/or derivatives thereof, at least one oligosaccharide and at least one inhibitor of hyaluronic acid degradation, it clearly increases the bioavailability of the hyaluronic acid, it makes it possible to space out the applications and to reduce the number thereof and it is highly effective in filling wrinkles, fine lines, fibroblast depletions and any scars.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be understood more clearly from the description to follow and the attached Figures of Drawing, in which:

FIG. 1 shows, in a semilogarithmic representation, the results of a study, the objective of which was to evaluate the inhibitory effect of glycyrrhizin on hyaluronidase activity of bovine origin, and

FIG. 2 shows the results of a study, the objective of which was to evaluate the effect of the combination “GLZ+pentamer (NAG-glucuronic acid)5+retinol” on the neosynthesis of hyaluronic acid by normal human keratinocytes.

DETAILED DESCRIPTION OF BEST MODE AND SPECIFIC/PREFERRED EMBODIMENTS OF THE INVENTION

The preparations according to the invention comprise, formulated into a physiologically acceptable medium, hyaluronic acid, and at least one retinoid and/or salts thereof and/or derivatives thereof, at least one oligosaccharide and at least one inhibitor of hyaluronic acid degradation.

The present invention thus also features products comprising:

hyaluronic acid,

at least one retinoid and/or salts thereof and/or derivatives thereof,

at least one oligosaccharide, preferably one, and

at least one inhibitor of hyaluronic acid degradation, preferably one, as a combination product for simultaneous, separate or sequential administration in the treatment of dermatological conditions.

Such a combination product is in particular effective in the treatment of wrinkles, fine lines, fibroblast depletions and scars.

The term “combination product” means a single composition comprising each of the active compounds, but also a complex composition comprising at least two different compositions, each comprising a part of the active agents of said preparation.

The term “active agents” means, according to the present invention, the compounds selected from hyaluronic acid, at least one oligosaccharide, at least one inhibitor of hyaluronic acid degradation and at least one retinoid and/or salts thereof and/or derivatives thereof. Thus, the combination product according to the invention comprises at least 4 active agents.

Exemplary combination products according to the invention include:

a single composition comprising hyaluronic acid, a retinoid and/or salts thereof and/or derivatives thereof, an oligosaccharide and an inhibitor of hyaluronic acid degradation;

a composition comprising just one of these four active agents, combined with a composition comprising at least the other three active agents;

a composition comprising two of these four active agents, combined with a composition comprising at least the other two active agents; and

a composition comprising three of these four active agents, combined with a composition comprising at least the fourth active agent.

Preferably, the combination product according to the invention comprises a composition A comprising hyaluronic acid in the form of an injectable (preferably aqueous) solution, combined with a composition B comprising:

at least one inhibitor of hyaluronic acid degradation, preferably glycyrrhizin,

at least one oligosaccharide, preferably the pentamer (NAC-glucuronic acid)5, and

at least one retinoid and/or salts thereof and/or derivatives thereof, preferably retinol,

in the form of a composition for topical application.

The term “physiologically acceptable medium” means, according to the invention, a medium compatible with the skin and, optionally, with its appendages (eyelashes, nails, hair) and/or the mucous membranes.

In the preparations according to the invention, the hyaluronic acid, the retinoid and/or salts thereof and/or derivatives thereof, the oligosaccharide and the inhibitor of hyaluronic acid degradation are present in proportions that can range from 0.0000001% to 10%, preferably from 0.00001% to 1% by weight, relative to the total weight of the preparation. In the present description, and unless otherwise specified, it is understood that, when concentration ranges are given, they include the upper and lower limits of said range.

The preparations according to the invention comprise hyaluronic acid.

The term “hyaluronic acid” means the compound constituted of the series of glucuronic acid and of N-acetylglucosamine.

Advantageously, the hyaluronic acid is natural.

The term “natural hyaluronic acid” means a hyaluronic acid that is non-stabilized and non-chemically modified in the form, in particular, of esters or amides or in the form of derivatives having “intra- and/or interchain bridges” (crosslinked), such modifications affecting the physicochemical characteristics and the biological properties of said hyaluronic acid, and also what becomes of it after administration.

The preparations according to the invention also comprise a retinoid and/or salts thereof and/or derivatives thereof, taken alone or as a mixture.

Among the retinoids that may be part of the preparations according to the invention, retinol, retinal and/or retinoic acid, and salts and derivatives thereof, taken alone or as a mixture, will preferably be selected, more preferably retinol.

The term “retinoid salt” means, in particular, an alkali metal salt, an alkaline-earth metal salt or an organic amine salt. The term “retinoid derivative” means in particular the esters, such as retinyl palmitate, retinyl acetate, retinyl stearate, retinyl oleate, retinyl propionate or else retinyl linoleate.

Advantageously, the retinoids included in the preparations according to the invention are retinoids that exist naturally in the human body.

The preparations according to the invention also comprise an oligosaccharide.

The term “oligosaccharide” means, in particular, any oligosaccharide which limits the penetration of hyaluronic acid into the cells of the skin, in particular the keratinocytes and the fibroblasts.

Among the oligosaccharides, taken alone or as a mixture, that may be part of the preparations according to the invention, hyaluronic acid oligomers, preferably hyaluronic acid trimers to decamers, more preferably hyaluronic acid tetramers to hexamers, more preferentially the hyaluronic acid pentamer, will be selected.

Advantageously, the oligosaccharides used in the preparations according to the invention are compounds that exist naturally in the human body.

In the preparations according to the invention, the oligosaccharide is used at concentrations of from 10⁻⁹ M and 10⁻³ M, preferably from 10⁻⁸ M and 10⁻⁵ M.

The preparations according to the invention also comprise an inhibitor of hyaluronic acid degradation.

The term “inhibitor of hyaluronic acid degradation” means a compound capable of reducing, or even blocking, either the extracellular or the intracellular catabolism of hyaluronic acid, preferably a compound capable of reducing, or even blocking, the extracellular catabolism of hyaluronic acid, more preferably a compound capable of inhibiting the extracellular hyaluronidase present in the skin.

Among the inhibitors of hyaluronic acid degradation, taken alone or as a mixture, that may be included in the preparations according to the invention, glycyrrhizin or glycyrrhetinic acid, and derivatives and/or analogs thereof, will in particular be selected.

Advantageously, the inhibitors of hyaluronic acid degradation included in the preparations according to the invention are natural.

In the preparations according to the invention, the inhibitor is present at concentrations of from 10⁻⁹ M and 10⁻² M, preferably from 10⁻⁶ M and 10⁻³ M.

The term “derivatives of glycyrrhizin or of glycyrrhetinic acid” means, in particular, the salts, the substituted derivatives, the enantiomers and the racemates of said compounds.

As salts of said compounds, exemplary are the salts obtained by addition of said compounds with an inorganic base, selected in particular from among sodium hydroxide, lithium hydroxide, calcium hydroxide, potassium hydroxide, magnesium hydroxide, ammonium hydroxide or zinc hydroxide, and alkali metal or alkaline-earth metal carbonates such as sodium, lithium, calcium, potassium, magnesium, ammonium or zinc carbonates and bicarbonates, or with an organic base, selected, in particular, from among methylamine, propylamine, trimethylamine, diethylamine, triethylamine, N,N-dimethylethanolamine, tris(hydroxymethyl)aminomethane, ethanolamine, pyridine, picoline, dicyclohexylamine, morpholine, procaine, lysine, arginine, histidine, N-methylglucamine or else phosphonium salts such as alkylphosphonium salts, arylphosphonium salts, alkylarylphosphonium salts, alkenylarylphosphoniums or quaternary ammonium salts such as tetra-n-butylammonium salts. Such salts are in particular the potassium salt of glycyrrhetinic acid, the sodium salt of glycyrrhetinic acid, or else the monoammonium salt of glycyrrhetinic acid (ammonium glycyrrhetinate).

The term “analog” means, in particular, the enzymatic or biomimetic analogs of said compounds, capable of binding to the catalytic or noncatalytic site of hyaluronidases and of thus inhibiting their activation. Such analogs may be selected, in vitro, by means of hyaluronidase binding or inhibition assays according to the techniques conventionally used.

Advantageously, the derivatives and/or analogs should be of natural origin.

The compounds and derivatives and/or analogs thereof of natural origin are compounds in the pure state or in solution at various concentrations, obtained by various methods for extracting or hydrolyzing biological material of natural origin.

In a known manner, the preparations according to the invention may also contain the usual adjuvants known to those skilled in the art.

The preparations according to the invention are formulated for topical and/or parenteral application.

When they are for topical application, the preparations may be in any of the galenical forms normally employed for topical administration.

Exemplary topical preparations include preparations in liquid, pasty or solid form, and more particularly in the form of ointments, aqueous, aqueous-alcoholic or oily solutions, dispersions of the optionally two-phase lotion type, serum, aqueous, anhydrous or lipophilic gels, powders, impregnated pads, syndets, wipes, sprays, foams, sticks, shampoos, compresses, washing bases, emulsions of liquid or semi-liquid consistency of the milk type, obtained by dispersion of a fatty phase in an aqueous phase (O/W) or vice versa (W/O), a microemulsion, suspensions or emulsions of soft, semi-liquid or solid consistency of the white or colored cream, gel or ointment type, suspensions of microspheres or nanospheres or lipid or polymeric vesicles, or microcapsules, microparticles or nanoparticles or polymeric or gelled patches for controlled release.

When they are for parenteral administration, the preparations according to the invention may be administered subcutaneously or intradermally. Exemplary parenteral preparations include preparations in the form of solutions or suspensions for perfusion or for injection.

According to the invention, the compounds constituting the preparation may be administered according to the same method of administration or according to a combined method of administration.

The term “combined method of administration” means the administration of one or more compound(s) of the preparation according to the invention by topical administration combined with a parenteral administration, in particular by subcutaneous or intradermal injection of the other compound(s) of the preparation.

Advantageously, one fraction of the compounds is first applied topically and another fraction of said compounds is then applied parenterally, or vice versa.

According to an alternative embodiment of the invention, it is also possible to simultaneously apply one fraction of the compounds topically and another fraction of said compounds parenterally.

Indeed, the hyaluronic acid may even be administered in the form of an injectable aqueous solution, the retinol, the hyaluronic acid pentamer and the glycyrrhizin being administered in the form of a cream.

In the context of a combined administration, the administration frequencies may be identical or different.

As one example, the frequency of administration of hyaluronic acid injected in the form of an injectable aqueous solution may range from 1 to 12 months, preferably from 6 to 12 months, whereas those of the other compounds of the preparation according to the invention, administered in the form of a cream, may range from 1 to 7 days, preferably from 1 to 3 days.

The process for the production a preparation according to the invention comprises a step of mixing an effective amount of hyaluronic acid, at least one retinoid and/or salts thereof and/or derivatives thereof, at least one oligosaccharide and at least one inhibitor of hyaluronic acid degradation. Preferably, said process comprises a step of preparing a physiologically acceptable medium, to which the active agents are added.

According to a specific embodiment of the invention, the process for the production of a preparation comprises the steps of preparing a physiologically acceptable medium and of mixing an effective amount of hyaluronic acid, retinol, hyaluronic acid pentamer, and glycyrrhizin and/or derivatives thereof and/or analogs thereof.

Advantageously, the process for the production of a combination product according to the invention comprises a first step of preparing an injectable solution, comprising mixing the hyaluronic acid with a physiologically acceptable medium, and a second step of preparing a formulation suitable for topical administration, comprising mixing at least one retinoid and/or salts thereof and/or derivatives thereof, with at least one oligosaccharide and at least one inhibitor of hyaluronic acid degradation in a physiologically acceptable medium.

The present invention also features administration of a preparation as described above as a medicament useful in the treatment and/or prevention of dermatological conditions/afflictions.

More particularly, this invention features administration of a preparation as described above as a medicament useful in the treatment of wrinkles, fine lines, fibroblast depletions and scars. Such a medicament is suitable for the treatment of wrinkled and/or aged skin, and is useful, in particular, to prevent and/or reduce the effects thereof. The treatment of wrinkles, fine lines, fibroblast depletions and any scars is carried out in particular by filling.

In particular, the preparations according to the invention may be applied to the areas of the face or of the forehead that are marked with expression wrinkles.

The present invention also features administration of a preparation as described above as a medicament useful in repair surgery.

In addition, this invention features such preparations within a dermal implant.

In order to further illustrate the present invention and the advantages thereof, the following specific examples are given, it being understood that same are intended only as illustrative and in nowise limitative. In said examples to follow, all parts and percentages are given by weight, unless otherwise indicated.

EXAMPLE 1 Inhibitory Effect of Glycyrrhizin (GLZ) on Hyaluronidase Activity of Bovine Origin Determination of the IC₅₀ of GLZ, With or Without Pre-Incubation at 37° C.

GLZ, at various concentrations, is or is not pre-incubated for 20 minutes at 37° C. in the presence of the enzyme. The enzyme reaction is triggered by adding the hyaluronic acid solution (time T0). After incubation for 20 minutes, the non-hydrolyzed hyaluronic acid is precipitated by adding acidic bovine albumin solution.

In order to verify that the pre-incubation step has no effect on the stability of the hyaluronidase, an aliquot of a solution of the enzyme is placed at 37° C. for 20 minutes. Another aliquot is conserved in an ice bath for 19 minutes, and is then incubated at 37° C. for 1 minute. A solution of hyaluronic acid is then added to each aliquot (T0). After incubation for 15, 30 or 45 minutes, the non-hydrolyzed hyaluronic acid is precipitated by addition of acidic bovine albumin solution.

Measurement of the Hyaluronidase Activity of Bovine Origin

After the precipitation step, the turbidimetry of the solutions is determined on a spectrophotometer at a wavelength of 600 nm. The optical density (OD) of these solutions is subtracted from the OD of a control solution of hyaluronic acid (of the same concentration) not hydrolyzed by the enzyme. This difference in OD, which is inversely proportional to the concentration of hyaluronic acid, is used to measure the activity of the hyaluronidase.

The inhibitory effect of GLZ on the bovine hyaluronidase is shown in FIG. 1 (semilogarithmic representation).

The results obtained show that this effect is dose-dependent and that the concentration of GLZ which gives 50% inhibition (IC₅₀) of the hyaluronidase activity is 400 μM without pre-incubation with the enzyme.

When the GLZ is pre-incubated for 20 minutes at 37° C. in the presence of the enzyme, the IC₅₀ is 350 μM.

Example 2 Inhibitory Effect of the “GLZ+Pentamer (NAG-Glucuronic acid)5+Retinol” Combination on the Neosynthesis of Hyaluronic Acid by Normal Human Keratinocytes

According to the prior art, it is accepted that an equilibrium pre-exists from the neosynthesis and the degradation of hyaluronic acid. In other words, the neosynthesis of hyaluronic acid is a reflection of its degradation: measuring the variations in one therefore amounts to measuring the variations in the other. For reasons of technical simplicity, the change in neosynthesis of hyaluronic acid in the presence of the “GLZ+pentamer (NAG-glucuronic acid)5+retinol” combination is measured, relative to the corresponding control.

The adult human keratinocytes (NHK) are isolated from a fragment of human skin collected after an abdominoplasty operation (subject CAOL, 38 years old).

The NHK are cultured to confluence as a monolayer in 24-well plates and sub-cultured. The NHK are used following the third passage.

The culture medium used is MCK medium at 37° C. in a humid atmosphere containing 5% CO₂.

The MCK culture medium is an SFM defined keratinocyte medium comprising growth factors, to which penicillin (50 IU/ml) and streptomycin (50 μg/ml) are added.

Preparation of Reagents

The pentamer (NAG-glucuronic acid)5 is dissolved at 2 mg/ml in the MIK culture medium (MCK medium containing 2 μCi/ml of radiolabeled glucosamine). It is then diluted in an MIK medium containing 0.2% DMSO.

This product is tested at 0.1-0.5 and 2 mg/ml.

The glycyrrhizin (GLZ) is dissolved at 400 mM in DMSO. It is then diluted in the MIK culture medium (MCK medium containing 2 μCi/ml of radiolabeled glucosamine).

This product is tested at 100-400 and 800 μM (concentration of DMSO kept constant: 0.2%).

The retinol is dissolved at 10 mM in DMSO. It is then diluted in the MIK culture medium (MCK medium containing 2 μCi/ml of radiolabeled glucosamine).

This product is tested at 1-10 and 100 nM (concentration of DMSO maintained constant: 0.2%).

The “GLZ+pentamer (NAG-glucuronic acid)5+retinol” combination is prepared by mixing the stock solutions prepared above. It is then diluted in MIK medium (MCK medium containing 2 μCi/ml of radiolabeled glucosamine).

This product is tested at:

100 μM GLZ+0.1 mg/ml pentamer (NAG-glucuronic acid)5+1 nM retinol;

400 μM GLZ+0.5 mg/ml pentamer (NAG-glucuronic acid)5+10 nM retinol;

800 μM GLZ+2 mg/ml pentamer (NAG-glucuronic acid)5+100 nM retinol.

These three solutions have a constant DMSO concentration of 0.2%.

Experimental protocol: (Pienimaki et al., The Journal of Biological Chemistry, (2001) Vol. 276, No. 23, June 8, p 20428-20435).

The reaction system is constituted of normal human keratinocytes as a monolayer culture in 24-well plates (cells at confluence at the beginning of the incubation).

The NHK are incubated in the presence of the test products and of radiolabeled glucosamine for 6-12-24 and 48 hours, in a final volume of 400 μl of medium per culture well. The incubation temperature is 37° C., the humid atmosphere contains 5% CO₂.

a. Recovery of All the GAGs Comprising Radiolabeled Glucosamine

At the end of the incubation, the culture media are recovered by centrifugation and then the cell layers are rinsed with PBS. The mixture composed of the rinsing medium and of the initial supernatant is recovered. After the addition of hyaluronic acid (10 μg/fraction, role of “carrier” during the precipitations with cetyl pyridinium chloride (CPC)) and of papain (200 μg/fraction, hydrolysis of proteoglycans with release of GAGs), the fractions are incubated for 2 hours at 60° C., and then the fractions are incubated for 10 minutes at 100° C. in order to inactivate the enzymes by heat denaturation. Finally, the GAGs are precipitated by the addition of CPC (final concentration of 1%) and incubated overnight at ambient temperature. After centrifugation for 10 minutes at 15 000 g, and then elimination of the supernatants containing the non-incorporated radiolabeled glucosamine, the pellets are rinsed with 1% CPC, and a further centrifugation of 10 minutes at 15 000 g is carried out, followed by elimination of the supernatants.

b. Measurement of Hyaluronic Acid Neosynthesis

The hyaluronic acid contained in the centrifugation pellets is specifically hydrolyzed by incubation (3 hours at 37° C.) of these pellets with hyaluronidase (hyaluronan lyase from Streptomyces hyalurolyticus, 0.25 unit/fraction). For this, the medium containing these pellets and the hyaluronidase are incubated for 3 hours at ambient temperature, followed by centrifugation for 10 minutes at 15 000 g. Finally, the non-hydrolyzed GAGs are precipitated by adding CPC (1% final concentration) in the presence of chondroitin sulfate (50 μg/fraction—role of “carrier”). The radioactivity of the supernatant, determined by liquid scintillation (β-counter), is directly proportional to the amount of hyaluronic acid neosynthesized during the incubation of the cells with the radiolabeled glucosamine.

The radioactivity measurements are given in the following tables:

TABLE 1 Measurement of hyaluronic acid neosynthesis by normal human keratinocytes in the absence of the “GLZ + pentamer (NAG- glucuronic acid)5 + retinol” combination (control). Incubation Incubation Incubation Incubation Incubation 6 h 12 h 24 h 48 h 72 h dpm 4156 12 216 38 404 62 938 64 701 3997 11 874 34 050 70 271 74 910 4232 12 463 30 264 62 411 79 646 Mean 4128 12 184 34 239 65 207 73 086 Standard  120 296 4073 4394 7638 deviation

TABLE 2 Measurement of hyaluronic acid neosynthesis by normal human keratinocytes in the presence of the “GLZ + pentamer (NAG-glucuronic acid)5 + retinol” combination. Incubation Incubation Incubation Incubation Incubation 6 h 12 h 24 h 48 h 72 h 100 μM GLZ + 0.1 mg/ml pentamer (NAG-glucuronic acid)5 + 1 nM retinol dpm 3508 12 844 28 289 51 974 63 822 3388 14 053 26 971 55 088 64 251 3697 13 347 30 200 56 652 61 504 Mean 3531 13 415  28 487**  54 571**  63 192** Standard  156 607 1624 2381 1478 deviation 400 μM GLZ + 0.5 mg/ml pentamer (NAG-glucuronic acid)5 + 10 nM retinol dpm 3672  9847 25 363 26 813 54 202 3698 10 724 27 404 36 235 49 799 3461 10 577 25 351 29 007 54 488 Mean 3610   10 383***   26 039***   30 685***   52 830*** Standard  130 470 1182 4930 2629 deviation 800 μM GLZ + 2 mg/ml pentamer (NAG-glucuronic acid)5 + 100 nM retinol dpm 3203 8263 17 640 32 714 33 910 3401 8875 19 682 30 070 29 693 2580 7590 21 320 29 092 29 194 Mean 3061    8243***   19 547***   30 625***   30 932*** Standard  428 643 1844 1874 2591 deviation **mean significantly different than that of the control group (p < 0.05) ***mean significantly different than that of the control group (p < 0.01)

The inhibitory effect of the “GLZ+pentamer (NAG-glucuronic acid)5+retinol” combination on the hyaluronic acid neosynthesis by normal human keratinocytes is shown in FIG. 2.

The results obtained show a marked inhibition of hyaluronic acid neosynthesis. This effect is dose-dependent and, at the highest concentrations of the three components, the inhibition of neosynthesis is of the order of 50% to 60%. Owing to the pre-existing equilibrium from neosynthesis and degradation of hyaluronic acid, this observed inhibition of neosynthesis corresponds to an equivalent inhibition of degradation.

One may therefore conclude that the effect of the combination protects hyaluronic acid against degradation. This system therefore makes it possible to increase the biostability of hyaluronic acid and, consequently, to improve its bioavailability.

Example 3 Composition No. 1 Injectable solution No. 1 Containing the 4 Components

This composition is prepared in a manner that is conventional for those skilled in the art:

Hyaluronic acid     2% Glycyrrhizin   0.02% Pentamer (NAC-glucuronic acid)5  0.002% Retinol 0.00001% Water qs 100%

Example 4 Composition No. 2 Injectable Solution No. 2 Containing Hyaluronic Acid, Coupled with a Cream Containing the Other 3 Components Injectable Solution

Hyaluronic acid 2% Water qs 100%

Cream

Glycyrrhizin   0.02% Pentamer (NAC-glucuronic acid)5  0.002% Retinol 0.00001% Stearic acid   3.00% Mixture of glyceryl monostearate   2.5% and PEG stearate (100 EO) PEG stearate (20 EO)   1.0% Cyclopentadimethylsiloxane  10.00% Plant oils   7.00% Synthetic oils   6.00% Silicone gum   0.20% Stearyl alcohol   1.00% Water qs 100%

Each patent, patent application, publication, text and literature article/report cited or indicated herein is hereby expressly incorporated by reference in its entirety.

While the invention has been described in terms of various specific and preferred embodiments, the skilled artisan will appreciate that various modifications, substitutions, omissions, and changes may be made without departing from the spirit thereof. Accordingly, it is intended that the scope of the present invention be limited solely by the scope of the following claims, including equivalents thereof. 

What is claimed is:
 1. A pharmaceutical/cosmetic composition which comprises a dermatologically effective amount of hyaluronic acid, at least one retinoid and/or salt and/or ester thereof, at least one oligosaccharide and at least one inhibitor of hyaluronic acid degradation, formulated into a physiologically acceptable medium therefor suitable for parenteral administration, said composition being effective in the treatment of wrinkles, fine lines, fibroblast depletions or scars.
 2. The pharmaceutical/cosmetic composition as defined by claim 1, said at least one retinoid and/or salt and/or ester thereof comprising retinol.
 3. The pharmaceutical/cosmetic composition as defined by claim 1, said at least one oligosaccharide comprising a hyaluronic acid oligomer.
 4. The pharmaceutical/cosmetic composition as defined by claim 1, said at least one inhibitor of hyaluronic acid degradation comprising glycyrrhizin or glycyrrhetinic acid, and/or salt thereof.
 5. The composition as defined by claim 1, comprising a solution or suspension for perfusion or for injection.
 6. A combination product comprising, in two fractions: (1) in a first fraction, a dermatologically effective amount of hyaluronic acid and zero to two further kinds of active agents selected from the group consisting of (a) at least one retinoid and/or salt and/or ester thereof, (b) at least one oligosaccharide and (c) at least one inhibitor of hyaluronic acid degradation, formulated into a physiologically acceptable medium therefor suitable for parenteral administration, and (2) in a second fraction, at least one of each kind of active agent defined in (a) through (c) above which is not present in said first fraction, formulated into a physiologically acceptable medium therefor suitable for topical application, for simultaneous, separate or sequential administration in the treatment of wrinkles, fine lines, fibroblast depletions or scars.
 7. The combination product as defined by claim 6, comprising a fraction A which comprises hyaluronic acid in the form of an injectable solution, and a fraction B which comprises at least one inhibitor of hyaluronic acid degradation, at least one oligosaccharide, and at least one retinoid and/or salt and/or ester thereof, formulated for topical application.
 8. A process for the production of a pharmaceutical/cosmetic composition as defined by claim 1, said process comprising mixing an effective amount of hyaluronic acid, at least one retinoid and/or salt and/or ester thereof, at least one oligosaccharide and at least one inhibitor of hyaluronic acid degradation, and formulating same into a physiologically acceptable medium therefor suitable for parenteral administration.
 9. A process for the production of a combination product as defined by claim 7, said process comprising: a first step of preparing an injectable solution, which comprises mixing the hyaluronic acid with a physiologically acceptable medium suitable for injection, and a second step of formulating a composition suitable for topical administration, comprising mixing at least one retinoid and/or salt and/or ester thereof with at least one oligosaccharide and at least one inhibitor of hyaluronic acid degradation in a physiologically acceptable medium therefor suitable for topical administration.
 10. A method for the treatment of wrinkles, fine lines, fibroblast depletions or scars, said method comprising parenterally administering to an individual in need of such treatment, a pharmaceutical/cosmetic composition as defined by claim
 1. 11. A method for conducting repair surgery on an individual in need of such treatment, said method comprising, before or during such repair surgery, parenterally administering to said individual a thus effective amount of a pharmaceutical/cosmetic composition as defined by claim
 1. 12. A method for the treatment of wrinkles, fine lines, fibroblast depletions or scars afflicting an individual's skin, said method comprising filling same by injection with a thus effective amount of a pharmaceutical/cosmetic composition as defined by claim
 1. 13. A method for the treatment of wrinkles, fine lines, fibroblast depletions or scars, said method comprising parenterally administering the first fraction of, and topically applying the second fraction of, the combination product as defined by claim 6 to an individual in need of such treatment.
 14. A method for conducting repair surgery on an individual in need of such treatment, said method comprising, before or during such surgery, parenterally administering the first fraction of, and topically applying the second fraction of, the combination product as defined by claim 6 to an individual in need of such treatment.
 15. A method for the treatment of wrinkles, fine lines, fibroblast depletions or scars afflicting an individual's skin, said method comprising filling same by injection with the first fraction of, and topically applying the second fraction of, the combination product as defined by claim 6 in a thus effective amount.
 16. The pharmaceutical/cosmetic composition as defined by claim 1, wherein said at least one inhibitor of hyaluronic acid degradation is glycyrrhizin.
 17. The combination product as defined by claim 6, wherein said at least one inhibitor of hyaluronic acid degradation is glycyrrhizin.
 18. The combination product as defined by claim 6, wherein said at least one retinoid and/or salt and/or ester thereof is retinol.
 19. The combination product as defined by claim 6, wherein said at least one oligosaccharide is a hyaluronic acid oligomer.
 20. The pharmaceutical/cosmetic composition as defined by claim 3, where said hyaluronic acid oligomer is a hyaluronic acid tetramer, pentamer or hexamer.
 21. The combination product as defined by claim 19, wherein said hyaluronic acid oligomer is a hyaluronic tetramer, pentamer or hexamer.
 22. The pharmaceutical/cosmetic composition as defined by claim 20, wherein said hyaluronic acid oligomer is the pentamer (NAC-glucuronic acid)5.
 23. The combination product as defined by claim 21, wherein said hyaluronic acid oligomer is the pentamer (NAC-glucuronic acid)5.
 24. The pharmaceutical/cosmetic composition as defined by claim 1, said composition comprising hyaluronic acid, retinol, glycyrrhizin and pentamer (NAC-glucuronic acid)5, formulated into a physiologically acceptable medium therefor suitable for parenteral administration.
 25. The combination product as defined by claim 6, said combination product comprising (1) in a first fraction, hyaluronic acid, formulated into a physiologically acceptable medium therefor suitable for parenteral administration; and (2) in a second fraction, retinol, glycyrrhizin and pentamer (NAC-glucuronic acid)5, formulated into a physiologically acceptable medium therefor suitable for topical administration. 